Method for analyzing microorganism

ABSTRACT

[OBJECT] To enable accurate identification of a specific serotype of Salmonella bacteria in a method for analyzing a microorganism using a MALDI-MS.[MEANS FOR SOLVING PROBLEM] The present invention is a method for analyzing a microorganism including an identification step for determining which of Abony and Pakistan which are two serotypes of Salmonella bacteria is contained in a sample which contains either Abony or Pakistan, based on the presence or absence of a peak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in a mass spectrum obtained by a mass spectrometric analysis of the sample, or a method for analyzing a microorganism including an identification step for determining which of Minnesota. Infantis and Brandenburg which are three serotypes of Salmonella bacteria is contained in a sample which contains Minnesota. Infantis or Brandenburg, based on the presence or absence of a peak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in a mass spectrum obtained by a mass spectrometric analysis of the sample, or a method for analyzing a microorganism including an identification step for determining which of Schwarzengrund and Montevideo which are two serotypes of Salmonella bacteria is contained in a sample which contains either Schwarzengrund or Montevideo, based on the presence or absence of a peak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in a mass spectrum obtained by a mass spectrometric analysis of the sample.

TECHNICAL FIELD

The present invention relates to a method for analyzing a microorganism.

BACKGROUND ART

A matrix assisted laser desorption/ionization method, which is one typeof ionization method in mass spectrometry, is an ionization method foranalyzing a substance that barely absorbs laser light, or a substancethat is likely to be damaged by laser light, such as proteins. A matrixsubstance, which considerably absorbs laser light and is thereby easilyionized, is mixed with a substance to be analyzed, and the mixture isirradiated with laser light to ionize the substance to be analyzed.Typically, the matrix substance is prepared in the form of a solutionand is mixed with the substance to be analyzed. The solvent in thesolution is subsequently vaporized to obtain a dried matrix in the formof a crystal containing the substance to be analyzed. Then, the mixtureis irradiated with laser light, whereby the matrix substance absorbs theenergy of the laser light and becomes rapidly heated to be ultimatelyvaporized. The substance to be analyzed is also vaporized with thematrix substance. Through this process, the substance to be analyzedbecomes ionized.

A mass spectrometer using such a MALDI method (MALDI-MS) can analyze ahigh-molecular compound, such as a protein, without causing asignificant dissociation of the compound, and is also suitable formicroanalysis. Therefore, this type of mass spectrometer has been widelyused in the area of life science. One application of the MALDI-MS in thearea of life science is the identification of microorganisms usingMALDI-MS. This is a method in which a microorganism is identified basedon a mass spectrum pattern obtained using a test microorganism. Sincethis method can provide analysis results within a short period of time,the identification of a microorganism can be conveniently and speedilyperformed.

For example, a representative causative organism of food poisoning isSalmonella, which is a group of rod-shaped gram-negative facultativelyanaerobic bacteria of the family Enterobacteriaceae. There are threespecies belonging to Salmonella: Salmonella (which is hereinafterabbreviated as “S.”) enterica, S. bongori and S. subterranea. S.enterica is further divided into six subspecies. Many of the pathogenicSalmonella causative of food poisoning belong to S. enterica subsp.enterica. This subspecies is further divided into a large number ofserotypes. Determining the species, subspecies and serotypes ofSalmonella bacteria is important for elucidating the infection route offood poisoning and preventing the infection. Therefore, in recent years,attempts have been made to identify Salmonella bacteria using MALDI-MS.

CITATION LIST

Non Patent Literature

-   Non Patent Literature 1: Applied Microbiology and Biotechnology,    Vol. 101, issue 23-24, pp. 8557-8569, 2017

SUMMARY OF INVENTION Technical Problem

Determination of the species, subspecies and serotypes of Salmonellabacteria by MALDI-MS is achieved by detecting a biomarker peak, i.e., apeak whose position (mass-to-charge ratio; m/z value) and height (peakintensity; mV value) in the mass spectrum vary among bacterial bodies ofdifferent species, subspecies and serotypes. For the identification ofmicroorganisms including bacteria, a protein peak is often used as abiomarker peak (see Non Patent Literature 1).

For many proteins in closely related microorganisms, it is normally thecase that a peak originating from the same protein appears at the samemass-to-charge ratio m/z or within a narrow range of mass-to-chargeratios. Therefore, for an accurate identification of Salmonella bacteriaat the level of serotype, it is not enough to select a peak originatingfrom one protein as a biomarker peak; it is necessary to select peaksoriginating from a plurality of appropriate kinds of proteins for theserotype concerned. Additionally, there are only a limited number ofserotypes which are known to be identifiable through the use ofbiomarker peaks. Identifying an even greater number of serotypes hasbeen desired.

For example, Non Patent Literature 1 discloses a method in which thevalues of the mass-to-charge ratios of peaks originating from 12 kindsof proteins are read from a mass spectrum acquired by a massspectrometric analysis of a sample containing microorganisms, and theserotype of Salmonella to which the microorganism contained in thesample belongs is determined from those values. However, in some cases,the use of the peaks originating from the 12 kinds of proteins as markerpeaks can merely show that the serotype of the microorganism is among aplurality of specific serotypes of Salmonella bacteria, withoutspecifically demonstrating which of those serotypes is the actualserotype.

The problem to be solved by the present invention is to enable accurateidentification of a specific serotype of Salmonella bacteria in a methodfor analyzing a microorganism using a MALDI-MS.

Solution to Problem

The present invention developed for solving the previously describedproblem provides a method for analyzing a microorganism including:

an identification step for determining which of Abony and Pakistan whichare two serotypes of Salmonella bacteria is contained in a sample whichcontains either Abony or Pakistan, based on the presence or absence of apeak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in amass spectrum obtained by a mass spectrometric analysis of the sample,

where the value or values of the predetermined mass-to-charge ratio orratios are selected from the group consisting of 3028, 3119, 4166, 5487,5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 7453,7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687,9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435,13444, 15714, 15803 and 15990 as well as any combination of thesevalues.

The present invention developed for solving the previously describedproblem also provides a method for analyzing a microorganism including:

an identification step for determining which of Minnesota, Infantis andBrandenburg which are three serotypes of Salmonella bacteria iscontained in a sample which contains Minnesota, Infantis or Brandenburg,based on the presence or absence of a peak (or peaks) at a predeterminedmass-to-charge ratio (or ratios) in a mass spectrum obtained by a massspectrometric analysis of the sample,

where the value or values of the predetermined mass-to-charge ratio orratios are selected from the group consisting of 6094, 6483, 6689, 6719,6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857,12209, 13367, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878,15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766 and 20838as well as any combination of these values.

The present invention developed for solving the previously describedproblem also provides a method for analyzing a microorganism including:

an identification step for determining which of Schwarzengrund andMontevideo which are two serotypes of Salmonella bacteria is containedin a sample which contains either Schwarzengrund or Montevideo, based onthe presence or absence of a peak (or peaks) at a predeterminedmass-to-charge ratio (or ratios) in a mass spectrum obtained by a massspectrometric analysis of the sample,

where the value or values of the predetermined mass-to-charge ratio orratios are selected from the group consisting of 5096, 6699, 6733, 6830,9034, 12262, 12276, 13074, 15820, 15835 and 19001 as well as anycombination of these values.

Advantageous Effects of Invention

According to the present invention, if the sample contains a kind ofbacteria which is Abony or Pakistan which are two serotypes ofSalmonella bacteria, it is possible to correctly determine which of thetwo aforementioned serotypes is the serotype of the bacteria. If thesample contains a kind of bacteria which is Minnesota, Infantis orBrandenburg which are three serotypes of Salmonella bacteria, it ispossible to correctly determine which of the three aforementionedserotypes is the serotype of the bacteria. If the sample contains a kindof bacteria which is Schwarzengrund or Montevideo which are twoserotypes of Salmonella bacteria, it is possible to correctly determinewhich of the two aforementioned serotypes is the serotype of thebacteria.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic overall configuration diagram of amicroorganism-analyzing system used for a method for analyzing amicroorganism according to the present invention.

FIG. 2 is a flowchart showing one example of the procedure of the methodfor analyzing a microorganism.

FIG. 3 shows mass spectra obtained for two samples which respectivelycontained bacterial bodies of Abony and Pakistan which are two serotypesof Salmonella bacteria (first group), with each mass spectrum includingone of the seven peaks shown in Table 2 which satisfy the secondcriterion, among the peaks in the mass spectra.

FIG. 4 shows mass spectra obtained for two samples which respectivelycontained bacterial bodies of Abony and Pakistan which are two serotypesof Salmonella bacteria (first group), with each mass spectrum includingone of the seven peaks shown in Table 2 which do not satisfy the secondcriterion but satisfy the first criterion, among the peaks in the massspectra.

FIG. 5 shows mass spectra obtained for three samples which respectivelycontained bacterial bodies of Minnesota, Infantis and Brandenburg whichare three serotypes of Salmonella bacteria (second group), with eachmass spectrum including one of the three peaks shown in Table 4 whichsatisfy the second criterion. among the peaks in the mass spectra.

FIG. 6 shows mass spectra obtained for three samples which respectivelycontained bacterial bodies of Minnesota, Infantis and Brandenburg whichare three serotypes of Salmonella bacteria (second group), with eachmass spectra including one of the three peaks shown in Table 3 which donot satisfy the second criterion but satisfy the first criterion, amongthe peaks in the mass spectra.

FIG. 7 shows mass spectra obtained for two samples which respectivelycontained bacterial bodies of Schwarzengrund and Montevideo which aretwo serotypes of Salmonella bacteria (third group), with each massspectrum including one of the four peaks shown in Table 6 which satisfythe second criterion, among the peaks in the mass spectra.

FIG. 8 shows mass spectra obtained for two samples which respectivelycontained bacterial bodies of Schwarzengrund and Montevideo which aretwo serotypes of Salmonella bacteria (third group), with each massspectrum including one of the three peaks shown in Table 5 which do notsatisfy the second criterion but satisfy the first criterion, among thepeaks in the mass spectra.

DESCRIPTION OF EMBODIMENTS

The target of the method for analyzing a microorganism according to thepresent invention is a sample about which it is previously known thatthe microorganisms contained in the sample include a kind of bacteriabelonging to one of the three groups of serotypes of Salmonella. Thethree groups of serotypes are: the group consisting of Abony andPakistan which are two serotypes (hereinafter called the first group);the group consisting of Minnesota, Infantis and Brandenburg which arethree serotypes (hereinafter called the second group); and the groupconsisting of Schwarzengrund and Montevideo which are two serotypes(hereinafter called the third group). That is to say, the target of theanalysis is (a) a sample about which it is previously known that thesample contains a kind of Salmonella bacteria belonging to the firstgroup but it is unknown which of Abony and Pakistan is the serotype ofthe bacteria, or (b) a sample about which it is previously known thatthe sample contains a kind of Salmonella bacteria belonging to thesecond group but it is unknown which of Minnesota, Infantis andBrandenburg is the serotype of the bacteria, or (c) a sample about whichit is previously known that the sample contains a kind of Salmonellabacteria belonging to the third group but it is unknown which ofSchwarzengrund and Montevideo is the serotype of the bacteria.

That is to say, a method for analyzing a microorganism according to thepresent invention is a method for analyzing a microorganism including:

an identification step for determining which of Abony and Pakistan whichare two serotypes of Salmonella bacteria is contained in a sample whichcontains either Abony or Pakistan, based on the presence or absence of apeak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in amass spectrum obtained by a mass spectrometric analysis of the sample,

where the value or values of the predetermined mass-to-charge ratio orratios are selected from the group consisting of 3028, 3119, 4166, 5487,5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 7453,7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687,9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435,13444, 15714, 15803 and 15990 as well as any combination of thesevalues. In particular, the value (or values) of the predeterminedmass-to-charge ratio or ratios should preferably be selected from thegroup consisting of 3119, 4166, 5487, 6238, 6720, 7858, 8330, 8536,8687, 9912, 12276, 15714, 15803 and 15990 as well as any combination ofthese values.

The state of the peak detection for Abony and Pakistan which are twoserotypes corresponding to the previously mentioned mass-to-chargeratios are shown in Tables 1 and 2. In the present invention, the peaksat the mass-to-charge ratios shown in Table 1 or 2 are used as markerpeaks, and which of the two serotypes Abony and Pakistan is the actualserotype is determined by checking the state of the detection of thosepeaks.

TABLE 1 Mass-to-Charge Ratio (m/z) S. Abony S. Pakistan 3028.3 ∘ x3119.4 ∘ x 4165.7 ∘ x 5487.4 x ∘ 5506.7 x ∘ 5924.3 ∘ x 6010.9 ∘ x 6095.3∘ x 6238.3 ∘ x 6261.2 x ∘ 6369.4 ∘ x 6719.7 ∘ x 6724.6 x ∘ 6933.3 x ∘7272.2 x ∘ 7453.4 ∘ x 7480.2 x ∘ 7588.5 ∘ x 7858.2 x ∘ 7902.9 ∘ x 8053.4x ∘ 8129.3 x ∘ 8329.5 ∘ x 8460.9 ∘ x 8536.2 ∘ x 8546.2 x ∘ 8633.7 ∘ x8687.2 ∘ x 9669.0 x ∘ 9911.9 ∘ x 10956.1 ∘ x 11498.9 x ∘ 11505.5 ∘ x11847.4 ∘ x 12276.2 ∘ x 13365.7 ∘ x 13372.6 x ∘ 13435.4 ∘ x 13444.3 x ∘15713.9 x ∘ 15803.3 ∘ x 15990.2 ∘ x

TABLE 2 Mass-to-Charge Ratio (m/z) S. Abony S. Pakistan 3119.4 ∘ x4165.7 ∘ x 5487.4 x ∘ 6238.3 ∘ x 6719.7 ∘ x 7858.2 x ∘ 8329.5 ∘ x 8536.2∘ x 8687.2 ∘ x 9911.9 ∘ x 12276.2 ∘ x 15713.9 x ∘ 15803.3 ∘ x 15990.2 ∘x

Another method for analyzing a microorganism according to the presentinvention is a method for analyzing a microorganism including:

an identification step for determining which of Minnesota, Infantis andBrandenburg which are three serotypes of Salmonella bacteria iscontained in a sample which contains Minnesota, Infantis or Brandenburg,based on the presence or absence of a peak (or peaks) at a predeterminedmass-to-charge ratio (or ratios) in a mass spectrum obtained by a massspectrometric analysis of the sample,

where the value or values of the predetermined mass-to-charge ratio orratios are selected from the group consisting of 6094, 6483, 6689, 6719,6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857,12209, 13367, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878,15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766 and 20838as well as any combination of these values. In particular, the value orvalues of the predetermined mass-to-charge ratio or ratios shouldpreferably be selected from the group consisting of 6483, 7940, 10990,11808, 11821, 11848, 11857, 12209, 13406, 13445, 15803, 15878, 15895,17713 and 17735 as well as any combination of these values.

The state of the peak detection for Minnesota, Infantis and Brandenburgwhich are three serotypes corresponding to the previously mentionedmass-to-charge ratios are shown in Tables 3 and 4. In the presentinvention, the peaks at the mass-to-charge ratios shown in Table 3 or 4are used as marker peaks, and which of the three serotypes Minnesota,Infantis and Brandenburg is the actual serotype is determined bychecking the state of the detection of those peaks.

TABLE 3 Mass-to-Charge S. Minnesota S. Infantis S. Brandenburg Ratio(m/z) I II I II I 6094.0 x x ∘ ∘ ∘ 6483.3 ∘ ∘ x ∘ ∘ 6688.5 ∘ ∘ x ∘ ∘6719.0 x x ∘ x x 6872.2 ∘ ∘ x ∘ ∘ 7857.5 ∘ x x x x 7939.5 x x ∘ x ∘7947.5 ∘ ∘ x ∘ x 9322.0 x x x ∘ ∘ 10667.0 x x x ∘ x 10990.3 x x ∘ x x11807.5 ∘ ∘ x ∘ ∘ 11821.4 x x ∘ x x 11847.8 ∘ ∘ x ∘ ∘ 11856.7 x x ∘ x x12209.3 x x ∘ x x 13366.5 ∘ x ∘ ∘ x 13376.0 x ∘ x x ∘ 13406.3 x x x ∘ x13445.2 ∘ ∘ x ∘ ∘ 13475.9 x ∘ x x ∘ 14882.1 x x x x ∘ 15715.6 ∘ x x x x15803.1 x x ∘ x x 15878.4 x x ∘ x ∘ 15894.7 ∘ ∘ x ∘ x 15991.1 x ∘ ∘ ∘ ∘17713.0 ∘ ∘ x ∘ ∘ 17734.5 x x ∘ x x 17812.8 ∘ ∘ x ∘ ∘ 17835.0 x x ∘ x x18972.3 x x ∘ ∘ ∘ 19127.0 x x ∘ x x 20765.8 ∘ ∘ ∘ x x 20837.6 x x x x ∘

TABLE 4 Mass-to-Charge S. Minnesota S. Infantis S. Brandenburg Ratio(m/z) I II I II I 6483.3 ∘ ∘ x ∘ ∘ 7939.5 x x ∘ x ∘ 10990.3 x x ∘ x x11807.5 ∘ ∘ x ∘ ∘ 11821.4 x x ∘ x x 11847.8 ∘ ∘ x ∘ ∘ 11856.7 x x ∘ x x12209.3 x x ∘ x x 13406.3 x x x ∘ x 13445.2 ∘ ∘ x ∘ ∘ 15803.1 x x ∘ x x15878.4 x x ∘ x ∘ 15894.7 ∘ ∘ x ∘ x 17713.0 ∘ ∘ x ∘ ∘ 17734.5 x x ∘ x x

Another method for analyzing a microorganism according to the presentinvention is a method for analyzing a microorganism including:

an identification step for determining which of Schwarzengrund andMontevideo which are two serotypes of Salmonella bacteria is containedin a sample which contains either Schwarzengrund or Montevideo, based onthe presence or absence of a peak (or peaks) at a predeterminedmass-to-charge ratio (or ratios) in a mass spectrum obtained by a massspectrometric analysis of the sample,

where the value or values of the predetermined mass-to-charge ratio orratios are selected from the group consisting of 5096, 6699, 6733, 6830,9034, 12262, 12276, 13074, 15820, 15835 and 19001 as well as anycombination of these values. In particular, the value or values of thepredetermined mass-to-charge ratio or ratios should preferably beselected from the group consisting of 5096, 6699, 6733, 6830, 9034,12276, 15820 and 15835 as well as any combination of these values.

The state of the peak detection for Schwarzengrund and Montevideo whichare two serotypes corresponding to the previously mentionedmass-to-charge ratios are shown in Tables 5 and 6. In the presentinvention, the peaks at the mass-to-charge ratios shown in Table 5 or 6are used as marker peaks, and which of the two serotypes Schwarzengrundand Montevideo is the actual serotype is determined by checking thestate of the detection of those peaks.

TABLE 5 Mass-to-Charge Ratio (m/z) S. Schwarzengrund S. Montevideo5096.4 x ∘ 6699.2 ∘ x 6733.1 x ∘ 6830.1 ∘ x 9034.4 ∘ x 12261.9 x ∘12276.1 ∘ x 13074.0 x ∘ 15819.5 ∘ x 15834.5 x ∘ 19001.0 x ∘

TABLE 6 Mass-to-Charge Ratio (m/z) S. Schwarzengrund S. Montevideo5096.4 x ∘ 6699.2 ∘ x 6733.1 x ∘ 6830.1 ∘ x 9034.4 ∘ x 12276.1 ∘ x15819.5 ∘ x 15834.5 x ∘

As for the criterion for the peak extraction in the previously describedmethods for analyzing a microorganism, either the first criterion orsecond criterion which will be described later may be used, although thecriteria are not limited to these examples. Since the second criterionapplies stricter conditions to the peak extraction than the first one,it is likely that using the second criterion decreases incorrectdeterminations. However, since the intensity ratio of the peaks maychange depending on the strain or culture conditions, it is preferableto decide the use of the first or second criterion according to thepurpose of the determination of the serotype. It is also possible toinitially extract peaks according to the first criterion and furtherextract peaks which satisfy the second criterion among the initiallyextracted peaks. It is also possible to initially extract peaksaccording to the second criterion, and if it is still difficult toidentify the target serotype, peaks which do not satisfy the secondcriterion but satisfy the first criterion may subsequently be extractedfor identification. The value of the mass-to-charge ratio of each peakshould be understood as a rough value and inclusive of a certain rangeof variation depending on the type of device for mass spectrometry,conditions of the analysis or other factors. Tables 1, 3 and 5 show thestates of the peak detection using the first criterion, while Tables 2,4 and 6 show the states of the peak detection using the secondcriterion. In each table, the circle indicates that a peak was detected,while the cross indicates that no peak was detected.

(1) First Criterion

In the case of a group consisting of two serotypes, a peak should beextracted (a) if the peak is detected with an S/N value equal to orgreater than three in a mass spectrum obtained from a sample containingone of the two serotypes of Salmonella bacteria, while the same peak ina mass spectrum obtained from a sample containing the other serotype ofSalmonella bacteria is not detected, or is detected with an S/N valuenot greater than one fifth of the S/N value of the first mass spectrum,or (b) if the peak in one mass spectrum is detected with an S/N valueequal to or greater than three, while the same peak in the other massspectrum is not detected, or is detected with an S/N value not greaterthan one tenth of the S/N value of the first mass spectrum and isaccompanied by, or partially overlaps with, a nearby peak having a closevalue of the mass-to-charge ratio m/z.

In the case of a group consisting of three serotypes, a peak should beextracted (a) if the peak is detected with an S/N value equal to orgreater than three in one of the three mass spectra obtained from threesamples respectively containing any one of the three serotypes ofSalmonella bacteria, while the same peak in the two other mass spectrais not detected, or is detected with an S/N value not greater than onefifth of the S/N value of the first mass spectrum, or (b) if the peak inone of the three mass spectra is detected with an S/N value equal to orgreater than three, while the same peak on the two other mass spectra isnot detected or is detected with an S/N value not greater than one tenthof the S/N value of the first mass spectrum and is accompanied by, orpartially overlaps with, a nearby peak having a close value of themass-to-charge ratio m/z.

(2) Second Criterion

In the case of a group consisting of two serotypes, a peak should beextracted if the peak is detected with an S/N value equal to or greaterthan ten in a mass spectrum obtained from a sample containing one of thetwo serotypes of Salmonella bacteria, while the same peak in a massspectrum obtained from a sample containing the other serotype ofSalmonella bacteria is not detected, or is detected with an S/N valuenot greater than one tenth of the S/N value of the first mass spectrum.

In the case of a group consisting of three serotypes, a peak should beextracted if the peak is detected with an S/N value equal to or greaterthan ten in one of the three mass spectra obtained from three samplesrespectively containing any one of the three serotypes of Salmonellabacteria, while the same peak in the two other mass spectra is notdetected, or is detected with an S/N value not greater than one tenth ofthe S/N value of the first mass spectrum.

In any case, a peak extracted based on the second criterion needs to bean isolated peak accompanied by neither a nearby peak nor partiallyoverlapping peak which leads to an incorrect determination.

In this case, the m/z value should be evaluated, for example, with anaccuracy of 800 ppm, or preferably 500 ppm. If a plurality of peaks arepresent within that accuracy, the peak having the closest m/z valueshould be selected as a marker peak.

The group to which a microorganism contained in a sample belongs amongthe first through third groups of Salmonella bacteria can be identified,for example, by using the values of the mass-to-charge ratios of thepeaks originating from the 12 kinds of proteins (gns, YaiA, YibT, PPI,L25, L21, S8, L17, L15, S7, YciF and SodA) disclosed in Non PatentLiterature.

In the method for analyzing a microorganism according to the presentinvention, the identification of one of the serotypes of bacteria ineach group may be based on the presence or absence of a peak at a singlemass-to-charge ratio. However, the identification accuracy will beimproved if the identification of one of the serotypes of bacteria ineach group is based on the presence or absence of the peaks at aplurality of mass-to-charge ratios.

As a mass spectrometer to be used for the method for analyzing amicroorganism according to the present invention, a mass spectrometerusing a matrix assisted laser desorption/ionization (MALDI) method(MALDI-MS) is preferable. As the MALDI-MS, a MALDI time-of-flight massspectrometer (MALDI-TOFMS) can preferably be used. Since the MALDI-MShas an extremely wide range of measurable mass-to-charge ratios, a massspectrum suited for an analysis of high-mass molecules, such as theproteins which are constituents of microorganisms, can be acquired.

Next, one embodiment of the microorganism-analyzing system to be usedfor the method for analyzing a microorganism according to the presentinvention is described.

FIG. 1 shows a schematic overall configuration of themicroorganism-analyzing system. This system is roughly divided into amass spectrometry unit 10 and a microorganism identification unit 20.The mass spectrometry unit 10 includes an ionization unit 11 configuredto ionize molecules or atoms in a sample by matrix assisted laserdesorption/ionization (MALDI) and a time-of-flight mass separator (TOF)12 configured to separate various ions, ejected from the ionization unit11, according to their mass-to-charge ratios.

The TOF 12 includes an extraction electrode 13 configured to extractions from the ionization unit 11 and guide them into an ion flight spacewithin the TOF 12, and a detector 14 configured to detect ions whichhave been mass-separated within the ion flight space.

The microorganism identification unit 20 is actually a workstation,personal computer or other types of computers, in which a centralprocessing unit (CPU) 21, memory 22, display unit 23 (e.g., a liquidcrystal display), input unit 24 (e.g., a keyboard and mouse), andstorage unit 30 consisting of a large-capacity storage (e.g., a harddisk drive or solid state drive) are connected to each other. Stored inthe storage unit 30 are an operating system (OS) 31, spectrum creationprogram 32, species determination program 33 and serotype determinationprogram 35 (a program according to the present invention), as well as afirst database 34 and second database 36. The microorganismidentification unit 20 further includes an interface (I/F) 25 forcontrolling a direct connection to an external device as well as aconnection with an external device through a local area network (LAN) orother types of networks. Through this interface 25, the microorganismidentification unit 20 is connected with the mass spectrometry unit 10by a network cable NW (or wireless LAN).

In FIG. 1 , a spectrum acquirer 37, m/z reader 38 and serotypeidentifier 39 are shown, being linked to the serotype determinationprogram 35. Each of those components is basically a functional meansimplemented at the software level by the CPU 21 executing the serotypedetermination program 35. The serotype determination program 35 does notneed to be an independent program. There is no specific limitation onits form; for example, it may be a built-in function of the speciesdetermination program 33 or that of a program for controlling the massspectrometry unit 10. As the species determination program 33, forexample, a program configured to identify microorganisms by aconventional fingerprinting method may be used.

In the configuration in FIG. 1 , the spectrum creation program 32,species determination program 33, serotype determination program 35,first database 34 and second database 36 are installed on a terminaldevice to be operated by users. Those components may be at leastpartially, or even entirely, installed on a separate device connectedwith the aforementioned terminal device via a computer network, with theseparate device configured to perform the processing by those programsand/or access to those databases according to commands from the terminaldevice.

The first database 34 in the storage unit 30 holds a large number ofmass lists related to known microorganisms. The mass list is a list ofthe mass-to-charge ratios of ions to be detected in a mass spectrometricanalysis of a specific kind of microorganic cell. Along with theinformation of the mass-to-charge ratios, the list additionally includesat least the information of the classifications (family, genus, species,etc.) to which the microorganic cell belongs (classificationinformation). Those mass lists should preferably be prepared based onactual measurement data obtained beforehand by actually performing massspectrometric analyses of various kinds of microorganic cells using thesame method for ionization and mass separation as used in the massspectrometry unit 10.

When the mass lists are to be prepared from the actual measurement data,the peaks which appear within a predetermined mass-to-charge-ratio rangeare initially extracted from mass spectra obtained as the actualmeasurement data. Peaks which mainly originate from proteins can beextracted by setting the aforementioned mass-to-charge-ratio range atapproximately 4000-30000, while unwanted peaks (noise) can be excludedby extracting each peak whose height (relative intensity) is equal to orhigher than a predetermined threshold. A list of the mass-to-chargeratios (m/z) of the extracted peaks is created for each kind of cell andrecorded in the first database 34 along with the aforementionedclassification information and other related information. In order toreduce the variation in gene expression due to the culture conditions,the microorganic cells to be used for collecting the actual measurementdata should preferably be cultured under previously normalizedconditions.

The second database 36 in the storage unit 30 holds informationconcerning marker proteins for identifying, known kinds ofmicroorganisms, by their serotypes which are classifications lower thanthe species. The information concerning the marker proteins includes atleast the information of the mass-to-charge ratios (m/z) of the markerproteins in the known kinds of microorganisms. The second database 36may also hold information concerning marker proteins for identifyingknown kinds of microorganisms by another sub-classification (e.g.,subspecies, pathotype or strain) other than the serotype, or by othercriteria.

The second database 36 in the present embodiment contains the values ofthe mass-to-charge ratios of peaks originating from 12 kinds of markerproteins (gns, YaiA, YibT, PPI, L25, L21, S8, L17, L15, S7, YciF andSodA) for determining the serotype of a test microorganism in the casewhere the microorganism is a kind of Salmonella bacteria (see Non PatentLiterature 1), as well as information related to marker proteins fordetermining the serotype of a test microorganism whose serotype has beenidentified as belonging to one of the first through third groups ofSalmonella bacteria. Specifically, this information includes the valuesof the mass-to-charge ratios of predetermined marker peaks fordetermining (a) which of the two serotypes belonging to the first groupis when the serotype of a test microorganism has been identified asbelonging to the first group, or (b) which of the three serotypesbelonging to the second group is when the serotype of a testmicroorganism has been identified as belonging to the second group, or(c) which of the two serotypes belonging to the third group is when theserotype of a test microorganism has been identified as belonging to thethird group (specifically, the combinations of the values of themass-to-charge ratios shown in Tables 1-6).

The values of the mass-to-charge ratios of the marker proteins stored inthe second database 36 should preferably be selected by comparing acalculation mass determined by translating the base sequence of eachmarker protein into an amino-acid sequence, and a mass-to-charge ratiodetected by an actual measurement. The base sequences of the markerproteins may be determined by sequencing method, or they may beretrieved from public databases, e.g., a database at NCBI (NationalCenter for Biotechnology Information). For the determination of thecalculation mass from the amino-acid sequence, the cutting of theN-terminal methionine residue should preferably be taken into account asa post-translational modification. Specifically, if the secondamino-acid residue to the last is Gly, Ala, Ser, Pro, Val, Thr or Cys,the theoretical value should be calculated on the assumption that theN-terminal methionine will be cut. Additionally, since the molecule tobe actually observed with a MALDI-TOF MS is in a protonated form, theaddition of the proton should also preferably be taken into account indetermining the calculation mass (i.e., a theoretical values of themass-to-charge ratio of ions to be obtained in an analysis of a proteinwith a MALDI-TOF MS).

Next, a procedure of the analysis of the serotype of Salmonella bacteriausing the previously described microorganism-analyzing system isdescribed with reference to the flowchart.

Initially, the user prepares a sample containing the constituents of atest microorganism, sets the sample in the mass spectrometry unit 10,and operates the same unit to perform the mass spectrometric analysis.The sample may be a cell extract, or cell constituents (e.g., ribosomalproteins) collected from the cell extract and purified. Bacterial bodiesor cell suspension may also be used as they are.

The spectrum creation program 32 receives detection signals from thedetector 14 via the interface 25, and creates a mass spectrum for thetest microorganism based on the detection signals (Step 101).

Next, the species determination program 33 compares the mass spectrum ofthe test microorganism with the mass lists of known microorganismsrecorded in the first database 34, and extracts a mass list of a knownmicroorganism having a similar pattern of mass-to-charge ratios to thatof the mass spectrum of the test microorganism, such as a mass listincluding a considerable number of peaks which coincide with those ofthe mass spectrum of the test microorganism within a predeterminedmargin of error (Step 102). The species determination program 33subsequently searches the first database 34 for the classificationinformation related to the mass list extracted in Step 102, to determinethe organism species to which the known microorganism corresponding tothe mass list belongs (Step 103). If the organism species is notSalmonella, the organism species is displayed on the display unit 23 asthe organism species of the test microorganism (Step 114), and theanalytical processing is completed. If the organism species isSalmonella, the analysis proceeds to the processing by the serotypedetermination program 35. In the case where it has been previouslydetermined by another method that the sample contains Salmonellabacteria, and the analysis can directly proceed to the serotypedetermination program 35 without using the species determination programusing the mass spectrum.

In the serotype determination program 35, the mass spectrum of the testmicroorganism is compared with the values of the mass-to-charge ratiosof the marker proteins recorded in the second database, to identify theserotype of the test microorganism (Step 104). Specifically, theserotype determiner 39 initially reads, from the second database 36, thevalues of the mass-to-charge ratios of the peaks originating from the 12aforementioned kinds of marker proteins (gns, YaiA, YibT, PPI, L25, L21,S8, L17, L15, S7, YciF and SodA). Subsequently, the spectrum acquirer 37obtains the mass spectrum of the test microorganism prepared in Step101. Then, for each marker protein, the m/z reader 38 selects acorresponding peak present in the mass spectrum within amass-to-charge-ratio range related to the marker protein in the seconddatabase 36, and determines the serotype based on the values of themass-to-charge ratios of the selected peaks.

If the determination result indicates that the serotype of the testmicroorganism is a serotype belonging to the first group (Abony orPakistan; Step 105), the values of the mass-to-charge ratios of thepredetermined marker peaks corresponding to the serotypes belonging tothe first group are retrieved from the second database (Step 109).

If the determination result indicates that the serotype of the testmicroorganism is a serotype belonging to the second group (Minnesota,Infantis or Brandenburg; Step 106), the values of the mass-to-chargeratios of the predetermined marker peaks corresponding to the serotypesbelonging to the second group are retrieved from the second database(Step 110).

If the determination result indicates that the serotype of the testmicroorganism is a serotype belonging to the third group (Schwarzengrundor Montevideo; Step 107), the values of the mass-to-charge ratios of thepredetermined marker peaks corresponding to the serotypes belonging tothe third group are retrieved from the second database (Step 111).

If the determination result indicates that the serotype of the testmicroorganism is a serotype which belongs to none of the first throughthird groups (Step 108), the serotype of the test microorganism is shownon the display unit 23 as a serotype which is none of the serotypesbelonging to the first through third groups (Step 114).

After the serotype has been identified as belonging to one of the firstthrough third groups, and the values of the mass-to-charge ratios of thepredetermined marker peaks corresponding to the serotypes belonging toeach group have been retrieved, the presence or absence of a peak ischecked for each of the mass-to-charge-ratio ranges related to thosevalues of the mass-to-charge ratios and stored in the second database 36(Step 112). Based on the state of the presence or absence of the peaks,the serotype of the test microorganism is determined (Step 113), and thedetermination is shown on the display unit 23 as the identificationresult for the test microorganism (Step 114).

EXAMPLE

Hereinafter described is an experiment conducted to prove the effect ofthe method for analyzing a microorganism according to the presentembodiment. It should be noted that the following descriptions aremerely illustrative and do not limit the present invention.

1. Culturing of Salmonella Bacteria

A total of seven kinds of Salmonella bacteria (Salmonella enterica),i.e., the two kinds of Salmonella bacteria belonging to the first group,the three kinds of Salmonella bacteria belonging to the second group,and the two kinds of Salmonella bacteria belonging to the third group,were cultured at 37 degrees Celsius for 20 hours using LB agar.

The kinds of Salmonella bacteria belonging to the first through thirdgroups are as follows.

(1) First Group

S. Abony, NBRC100797

S. Pakistan, GTC09493

(2) Second Group

S. Minnesota, NBRC15182

S. Infantis, ATCC BAA-1675

S. Brandenburg, jfriSe1402-3

(3) Third Group

S. Schwarzengrund, HyogoSO12004

S. Montevideo, jfriSe1409-6

2. Preparation of Matrix Solutions

The following two kinds of matrix solutions were prepared.

(2-1) Sinapine acid (SA) as the matrix substance was dissolved inethanol to obtain a matrix solution (saturated solution) with an SAcontent of 25 mg/mL. This matrix solution is hereinafter called “SA-1”.

(2-2) SA, methylene diphosphonate (MDPNA) and decyl-β-D-maltopyranoside(DMP) as a surfactant were dissolved in an aqueous solution with anacetonitrile (ACN) content of 50% and trifluoroacetic acid (TFA) contentof 0.6% to obtain a matrix solution with an SA content of 25 mg/mL,MDPNA content of 1%, and DMP content of 1 mM. This matrix solution ishereinafter called “SA-2”.

The SA used for the matrix solutions SA-1 and SA-2 was a product ofFUJIFILM Wako Pure Chemical Corporation. The MDPNA and DMP were productsof Sigma-Aldrich Japan LLC.

3. Preparation of Matrix-Microorganism Suspension

(3-1) From each of the 7 kinds of Salmonella bacteria cultured on LBagar, approximately 1 mg of sample was collected using a microbalanceand put in a tube. The matrix solution SA-2 was added to the sample inthe tube to obtain a solution with a bacteria concentration of 1mg/0.075 mL (1×10⁷ CFU/μL), and this solution was suspended with aneedle.(3-2) Ultrasonic vibrations were applied to the tube for one minute. Theobtained suspension was centrifuged (at 12000 rpm for 5 minutes) toobtain a centrifugation supernatant.4. Analysis with MALDI-MS(4-1) The matrix solution SA-1 was dropped into the wells on a MALDIsample plate, at 0.5 μL per one well (precoating).(4-2) Subsequently, the centrifugation supernatant was dropped into thewells precoated with the matrix solution SA-1, at 1 μL per one well, andwas let to naturally dry.(4-3) The MALDI sample plate obtained in (4-2) was set in a MALDI-MS(AXIMA Performance, manufactured by Shimadzu Corporation), and themeasurement was performed in a linear mode (positive ion mode). Allmeasurement data were acquired by a raster analysis. Raster analysis isan automatic measurement function provided in the previously mentionedmass spectrometer. In this technique, the sample in each well of thesample plate is irradiated with a predetermined number of laser shots ata predetermined number of points, to acquire mass spectrum data.5. Extraction of Peaks

A self-calibration of Salmonella bacteria was applied to the measurementdata (more specifically, a calibration process was performed using, asinternal standards, some peaks that have already been assigned tospecific Salmonella bacteria), and useful peaks for the identificationof the serotype were extracted from the obtained mass spectrum. Thevalues of the mass-to-charge ratios of the peaks originating from the 12kinds of marker proteins shown in Non Patent Literature 1 were read fromthe extracted peaks, and the serotype of Salmonella bacteria to whichthe microorganism contained in the sample belonged was determined fromthe read values.

In the determination of the serotype using the 12 kinds of markerproteins, when it was determined that the microorganism was either Abonyor Pakistan which are two serotypes of Salmonella bacteria, the state ofthe peak detection on the obtained mass spectrum was checked at one ormore values of the mass-to-charge ratios selected from the groupconsisting of 3028, 3119, 4166, 5487, 5507, 5924, 6011, 6095, 6238,6261, 6369, 6720, 6725, 6933, 7272, 7453, 7480, 7589, 7858, 7903, 8053,8129, 8330, 8461, 8536, 8546, 8634, 8687, 9669, 9912, 10956, 11499,11506, 11847, 12276, 13366, 13373, 13435, 13444, 15714, 15803 and 15990.

In the determination of the serotype using the 12 kinds of markerproteins, when it was determined that the microorganism was one ofMinnesota, Infantis and Brandenburg which are three serotypes ofSalmonella bacteria, the state of the peak detection on the obtainedmass spectrum was checked at one or more values of the mass-to-chargeratios selected from the group consisting of 6094, 6483, 6689, 6719,6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857,12209, 13367, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878,15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766 and 20838.

In the determination of the serotype using the 12 kinds of markerproteins, when it was determined that the microorganism was eitherSchwarzengrund or Montevideo which are two serotypes of Salmonellabacteria, the state of the peak detection on the obtained mass spectrumwas checked at one or more values of the mass-to-charge ratios selectedfrom the group consisting of 5096, 6699, 6733, 6830, 9034, 12262, 12276,13074, 15820, 15835 and 19001.

The m/z values were evaluated with an accuracy of 800 ppm, or preferably500 ppm. When a plurality of peaks were present within that accuracy,the peak having the closest m/z value was selected as the marker peak.

6. Results

(1) First Group

The state of the peak detection at the previously listed mass-to-chargeratios (m/z values) was checked on the mass spectra obtained from twosamples which respectively contained one of the two kinds of Salmonellabacteria belonging to the first group (S. Abony and S. Pakistan). Thestate of the detection was as shown in Tables 1 and 2. In Tables 1 and2, the circle indicates that the marker peak at the mass-to-charge ratioconcerned was detected, while the cross indicates that the marker peakwas not detected.

FIG. 3 shows the mass spectra, where each mass spectrum covers an m/zrange including 4165.7, 5487.4, 6238.3, 7858.2, 8329.5, 9911.9 or15713.9 among the m/z values of the peaks which satisfy the secondcriterion (i.e., the m/z values shown in Table 2). FIG. 4 shows the massspectra, where each mass spectrum covers an m/z range including 5506.7,6261.2, 7588.5, 9669.0, 11847.4, 13444.3 or 15990.2 among the m/z valuesof the peaks which satisfy the first criterion but do not satisfy thesecond criterion. In both FIG. 3 and FIG. 4 , the upper shows the massspectrum for S. Abony, and the lower shows the mass spectrum for S.Pakistan. The arrows in FIGS. 3 and 4 indicate the extracted peaks.

As shown in FIGS. 3 and 4 , each of the peaks extracted based on thefirst and second criteria is only present in either the mass spectrumfor S. Abony or the mass spectrum for S. Pakistan. Therefore, which ofS. Abony and S. Pakistan is the serotype of the Salmonella bacteriacontained in the sample can be determined by the presence or absence ofthe peak. That is to say, the serotype of the Salmonella bacteria can beidentified. In particular, the use the peaks which satisfy the secondcriterion enables a more correct identification of the serotype sincethose peaks have higher S/N ratios than the peaks which satisfy only thefirst criterion, and since those peaks have no nearby peaks. It shouldbe noted that any one of the peaks at the 42 mass-to-charge ratios (m/zvalues) shown in Table 1 can be solely used as a marker peak foridentifying the serotype of the Salmonella bacteria contained in asample, although using peaks at a plurality of mass-to-charge ratios(m/z values) as marker peaks enables a more accurate identification ofthe serotype of the Salmonella bacteria contained in a sample.

Thus, which of the two serotypes of Salmonella bacteria belonging to thefirst group (S. Abony and S. Pakistan) was the actual serotype could bedetermined by checking the state of the peak detection at themass-to-charge ratios (m/z values) in Table 1 or 2.

(2) Second Group

The state of the peak detection at the previously listed mass-to-chargeratios (m/z values) was checked on the mass spectra obtained from threesamples which respectively contained any one of the three kinds ofSalmonella bacteria belonging to the second group (S. Minnesota, S.Infantis and S. Brandenburg). The state of the detection was as shown inTables 3 and 4.

FIG. 5 shows the mass spectra, where each mass spectrum covers an m/zrange including 7939.5, 13406.3 or 15878.4/15894.7 among the m/z valuesof the peaks which satisfy the second criterion (i.e., the m/z valuesshown in Table 4).

FIG. 6 shows the mass spectra, where each mass spectrum covers an m/zrange including 7947.5, 9322.0 or 13366.5/13376.0 among themass-to-charge ratios (m/z values) of the peaks which satisfy the firstcriterion but do not satisfy the second criterion. In both FIG. 5 andFIG. 6 , the upper shows the mass spectrum obtained for the samplecontaining S. Minnesota, the middle shows the mass spectrum obtained forthe sample containing S. Infantis, and the lower shows the mass spectrumobtained for the sample containing S. Brandenburg. The arrows in FIGS. 5and 6 indicate the extracted peaks.

As shown in FIGS. 5 and 6 , each of the peaks extracted based on thefirst and second criteria is present or absent on only one of the massspectra obtained from the three samples which respectively contained S.Minnesota, S. Infantis and S. Brandenburg. Therefore, which of S.Minnesota, S. Infantis and S. Brandenburg is the serotype of theSalmonella bacteria contained in the sample can be determined by thepresence or absence of the peak. That is to say, the serotype of theSalmonella bacteria can be identified. In particular, the use the peakswhich satisfy the second criterion enables a more correct identificationof the serotype since those peaks have higher S/N ratios than the peakswhich satisfy only the first criterion, and since those peaks have nonearby peaks. It should be noted that any one of the peaks at the 35mass-to-charge ratios (m/z values) shown in Table 3 can be solely usedas a marker peak for identifying the serotype of the Salmonella bacteriacontained in a sample, although using peaks at a plurality ofmass-to-charge ratios (m/z values) as marker peaks enables a moreaccurate identification of the serotype of the Salmonella bacteriacontained in a sample.

Thus, which of the three serotypes of Salmonella bacteria belonging tothe second group (S. Minnesota, S. Infantis and S. Brandenburg) was theactual serotype could be determined by checking the state of the peakdetection at the mass-to-charge ratios (m/z values) in Table 3 or 4.

(3) Third Group

The state of the peak detection at the previously listed mass-to-chargeratios (m/z values) was checked on the mass spectra obtained from twosamples which respectively contained one of the two kinds of Salmonellabacteria belonging to the third group (S. Schwarzengrund and S.Montevideo). The state of the detection was as shown in FIGS. 7 and 8 .

FIG. 7 shows the mass spectra, where each mass spectrum covers an m/zrange including 5096.4, 6830.8, 12276.1 or 15834.5 among the m/z valuesof the peaks which satisfy the second criterion (i.e., the m/z valuesshown in Table 6).

FIG. 8 shows the mass spectra, where each mass spectrum covers an m/zrange including 12261.9, 13074.0 or 19001.0 among the m/z values of thepeaks which satisfy the first criterion but do not satisfy the secondcriterion. In both FIG. 7 and FIG. 8 , the upper shows the mass spectrumobtained for the sample containing S. Schwarzengrund, and the lowershows the mass spectrum obtained for the sample containing S.Montevideo. The arrows in FIGS. 7 and 8 indicate the extracted peaks.

As shown in FIGS. 7 and 8 , each of the peaks extracted based on thefirst and second criteria is only present on either the mass spectrum ofthe sample containing S. Schwarzengrund or the mass spectrum of thesample containing S. Montevideo. Therefore, which of S. Schwarzengrundand S. Montevideo is the serotype of the Salmonella bacteria containedin the sample can be determined by the presence or absence of the peak.That is to say, the serotype of the Salmonella bacteria can beidentified. In particular, the use the peaks which satisfy the secondcriterion enables a more correct identification of the serotype sincethose peaks have higher S/N ratios than the peaks which satisfy only thefirst criterion, and since those peaks have no nearby peaks. It shouldbe noted that any one of the peaks at the 11 mass-to-charge ratio (m/z)values shown in Table 5 can be solely used as a marker peak foridentifying the serotype of the Salmonella bacteria contained in asample, although using peaks at a plurality of mass-to-charge ratio(m/z) values as marker peaks enables a more accurate identification ofthe serotype of the Salmonella bacteria contained in a sample.

Thus, which of the two serotypes of Salmonella bacteria belonging to thethird group (S. Schwarzengrund and S. Montevideo) was the actualserotype could be determined by checking the state of the peak detectionat the mass-to-charge ratios (m/z values) in Table 5 or 6.

Modes of Invention

A person skilled in the art can understand that the previously describedillustrative embodiments are specific examples of the following modes.

(Clause 1) A method for analyzing a microorganism according to one modeis a method for analyzing a microorganism including:

an identification step for determining which of Abony and Pakistan whichare two serotypes of Salmonella bacteria is contained in a sample whichcontains either Abony or Pakistan, based on the presence or absence of apeak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in amass spectrum obtained by a mass spectrometric analysis of the sample,

where the value or values of the predetermined mass-to-charge ratio orratios are selected from the group consisting of 3028, 3119, 4166, 5487,5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 7453,7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687,9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435,13444, 15714, 15803 and 15990 as well as any combination of thesevalues.

(Clause 2) In the method for analyzing a microorganism described inClause 1, the value or values of the predetermined mass-to-charge ratioor ratios are specifically selected from the group consisting of 3119,4166, 5487, 6238, 6720, 7858, 8330, 8536, 8687, 9912, 12276, 15714,15803 and 15990 as well as any combination of these values.

According to the method for analyzing a microorganism described inClause 1, if it is previously known that the serotype of the Salmonellabacteria contained in a sample is one of the two serotypes (Abony andPakistan), it is possible to determine which of Abony and Pakistan isthe actual serotype. According to the method for analyzing amicroorganism described in Clause 2, which of Abony and Pakistan is theactual serotype can be more correctly determined.

(Clause 3) A method for analyzing a microorganism according to anothermode is a method for analyzing a microorganism including:

an identification step for determining which of Minnesota, Infantis andBrandenburg which are three serotypes of Salmonella bacteria iscontained in a sample which contains Minnesota, Infantis or Brandenburg,based on the presence or absence of a peak (or peaks) at a predeterminedmass-to-charge ratio (or ratios) in a mass spectrum obtained by a massspectrometric analysis of the sample,

where the value or values of the predetermined mass-to-charge ratio orratios are selected from the group consisting of 6094, 6483, 6689, 6719,6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857,12209, 13366, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878,15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766 and 20838as well as any combination of these values.

(Clause 4) In the method for analyzing a microorganism described inClause 3, the value or values of the predetermined mass-to-charge ratioor ratios are specifically selected from the group consisting of 6483,7940, 10990, 11808, 11821, 11848, 11857, 12209, 13406, 13445, 15803,15878, 15895, 17713 and 17735.

According to the method for analyzing a microorganism described inClause 3, if it is previously known that the serotype of the Salmonellabacteria contained in a sample is one of the three serotypes (Minnesota,Infantis and Brandenburg), it is possible to determine which ofMinnesota, Infantis and Brandenburg is the actual serotype. According tothe method for analyzing a microorganism described in Clause 4, which ofMinnesota, Infantis and Brandenburg is the actual serotype can be morecorrectly determined.

(Clause 5) A method for analyzing a microorganism according to stillanother mode is a method for analyzing a microorganism including:

an identification step for determining which of Schwarzengrund andMontevideo which are two serotypes of Salmonella bacteria is containedin a sample which contains either Schwarzengrund or Montevideo, based onthe presence or absence of a peak (or peaks) at a predeterminedmass-to-charge ratio (or ratios) in a mass spectrum obtained by a massspectrometric analysis of the sample,

where the value or values of the predetermined mass-to-charge ratio orratios are selected from the group consisting of 5096, 6699, 6733, 6830,9034, 12262, 12276, 13074, 15820, 15835 and 19001 as well as anycombination of these values.

(Clause 6) In the method for analyzing a microorganism described inClause 5, the value or values of the predetermined mass-to-charge ratioor ratios are specifically selected from the group consisting of 5096,6699, 6733, 6830, 9034, 12276, 15820 and 15835 as well as anycombination of these values.

According to the method for analyzing a microorganism described inClause 5, if it is previously known that the serotype of the Salmonellabacteria contained in a sample is one of the two serotypes(Schwarzengrund and Montevideo), it is possible to determine which ofSchwarzengrund and Montevideo is the actual serotype. According to themethod for analyzing a microorganism described in Clause 6, which ofSchwarzengrund and Montevideo is the actual serotype can be morecorrectly determined.

(Clause 7) The second mode is a program for making a computer executethe step described in one of Clauses 1-6.

The invention claimed is:
 1. A method for analyzing a microorganism,comprising: an identification step for determining which of Abony andPakistan which are two serotypes of Salmonella bacteria is contained ina sample which contains either Abony or Pakistan, based on presence orabsence of a peak or peaks at a predetermined mass-to-charge ratio orratios, in a mass spectrum obtained by a mass spectrometric analysis ofthe sample, where a value or values of the predetermined mass-to-chargeratio or ratios are selected from the group consisting of 3028, 3119,4166, 5487, 5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933,7272, 7453, 7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546,8634, 8687, 9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373,13435, 13444, 15714, 15803 and 15990 as well as any combination of thesevalues.
 2. The method for analyzing a microorganism according to claim1, wherein the value or values of the predetermined mass-to-charge ratioor ratios are selected from the group consisting of 3119, 4166, 5487,6238, 6720, 7858, 8330, 8536, 8687, 9912, 12276, 15714, 15803 and 15990.3. A non-transitory computer readable medium recording a program formaking a computer execute the step described in claim
 1. 4. The methodfor analyzing a microorganism according to claim 1, further comprising:a determination step for determining that a serotype of themicroorganism contained in the sample is either Abony or Pakistan basedon presence or absence of peak or peaks derived from gns, YaiA, YibT,PPI, L25, L21, S8, L17, L15, S7, YciF, or SodA as marker proteins in amass spectrum obtained by a mass spectrometric analysis of the sample,wherein the identification step is performed after the determinationstep.